Raman spectroscopy is an analytical technique providing molecule-specific information about a sample. When monochromatic light or radiation strikes material (generally in this document, “incident radiation”), the material (collectively, “sample”) will interact with the light. The sample may absorb part of the radiation energy and be raised to an excited electronic state; the sample also may lose part of the absorbed energy through non-radiative relaxation, and may revert to a lower electronic state while releasing reduced energy radiation in the form of fluorescence. A portion of the incident radiation also may be scattered by the sample. Scattered radiation may contain both an elastic component, in which radiation frequencies remain unchanged, and inelastic components with altered frequencies. Elastically scattered components are called Rayleigh scattering; inelastic components, if it is caused by light interacting with the vibrations of molecular bonds, is called Raman scattering.
The frequencies of Raman scattered emissions differ from the incident radiation by the amount of a single, multiple, or combinations of the same or different vibrational frequencies. The amount of the frequency differences is called the “Raman shift,” a characteristic of a molecule. The Raman shift, therefore, is useful in analyzing qualitative and quantitative characteristics of a sample. Raman spectra typically contain multiple narrow peaks specific to the chemical identity of a sample, and accordingly can be used in many applications requiring molecular specificity.
Several types of Raman spectroscopic systems have been developed, as well as methods for applying Raman techniques to sample analysis. Exemplary apparatus and methods for using Raman analyses techniques are disclosed in U.S. Pat. No. 6,141,095 issued Oct. 31, 2000 to Allen, et al., U.S. Pat. No. 6,281,971 B1 issued Aug. 28, 2001 to Allen, et al., and U.S. Pat. No. 6,353,476 B1 issued Mar. 5, 2002 to Allen, et al., which references are incorporated into this document.
Typically, a Raman device for spectral analysis contains at least four basic modules: an excitation source, optics for beam steering and signal collection, a spectral analyzer, and a detector. Modern instruments typically use lasers for excitation to provide a wide selection of wavelengths ranging from ultra-violet to near-IR. A spectral analyzer generally decomposes a Raman signal into many constituent frequencies for analysis. A dispersive analyzer, for example, uses a wavelength-dispersing element, such as a grating or prism, to separate different wavelengths. An FT-Raman analyzer may use an interferometer to generate an interferogram from a signal, and transform the signal into the frequency domain through a mathematical procedure. A relatively new form of analyzer uses a tunable filter to pass one frequency at a time, such as an acousto-optic tunable filter, or a liquid crystal tunable filter. Detectors commonly used for Raman spectroscopy include single detectors such as photo multiplier tubes for monochromators working in the visible region, InGaAs or cooled Ge detectors for Fourier Transform (“FT”) Raman using near-infrared excitations, multi-channel sensors such as charge coupled devices for spectrographs and imaging spectral analyzers working in the visible and ultraviolet region, and thermal focal plane array detectors in the near-infrared region.
Optics for Raman include laser band pass filters for purifying the monochromatic source, laser rejection filters for removing Rayleigh scattered components before sending a signal to a spectral analyzer, and optics for focusing an excitation beam onto the sample and collecting scattered light from the sample. In the most common configuration, which is called the back-scattering or epi-configuration, the same optics performs both focusing and collecting functions. For examination of remote samples, both the excitation and the signal may be carried through optical fibers over long distances.
Raman microscopy gained popularity during the last decade because of its capability to analyze microscopic samples down to the size of the sub-μm level. In a Raman microscope, the excitation beam is guided into and the signal beam from an objective lens that serves as focusing and collecting optics.
Until now, existing Raman microscopes using dispersive or FT analyzers are designed for use of a microscope that was an attachment to the spectrometer because existing research grade spectral analyzers are typically heavy and bulky. The current invention reverses that trend, and provides a compact Raman spectrometer that may be assembled as an attachment that may be mounted onto a variety of commercially available infinity corrected light microscopes. Instead of treating the microscope as an observation tool for the spectrometer, the Raman spectrometer disclosed and claimed in this document is an accessory or attachment for a microscope, allowing a user to perform spectral analysis on a sample through a microscope. The Raman spectrometer disclosed in this document is designed to accommodate the perspective, desires, and needs of microscopy practitioners, instead of Raman spectrocopists.
Many existing Raman systems are heavy and bulky because they are not designed specifically for microscopy. Their Optical components often have large apertures for high sensitivity, translating into large and heavy components and systems. A compact, and light-weight attachment is achieved by recognizing the unique feature of microscopes: their objective lenses have small apertures. A large optical aperture is not required to capture the signal from an objectives lens. Therefore, smaller optics may be used to achieve reduction in size, weight, and cost. Further reductions in the size, weight and cost due to the laser is possible by the method and apparatus disclosed in U.S. Pat. No. 6,141,095 issued to Allen et al., providing for use of standard diode lasers without frequency stabilization by measuring laser frequencies simultaneously with the Raman. Diode lasers are smaller and less expensive than other lasers.
Another aspect of the Raman spectroscope disclosed and claimed in this document is a means for introducing the laser beam into, and rejecting Rayleigh scattered radiation from, a Raman beam path using edge filters. Edge filters now known in the art typically are interference filters, not holographic notch filters. As will be appreciated by those skilled in the art, to use a back scattering configuration the excitation beam should be introduced into a Raman signal beam path prior to the focusing and collection optics. Thus, the excitation beam and the Raman signal beam should be combined into a common or the same path. Some have suggested such beam combining be achieved using beam splitters, aperture sharing optics, or dichroic filters at 45 degree incident angles. However, conventional beam splitters are inefficient, and aperture sharing is only suitable for collection optics with large apertures. When aperture size approaches that of the laser beam, however, the through-put of aperture sharing becomes very low. Some have suggested overcoming this problem by using a dichroic filter. However, at high incident angles, a dichroic filter is sensitive to the polarization state of light beams, and makes difficult the observation of Raman bands close to the laser line.
Beam-combining optics will reject Rayleigh components to some extent, but the major part of laser rejection may be achieved using interference edge filters and holographic notch filters at near normal incidence, located in the Raman spectrometer between a beam-combiner and the spectral analyzer. Interference filters used for laser rejection may be categorized into two types, edge filters having a wide spectral rejection range, and rugate notch filters having a narrow spectral rejection range. Both types are made of multi-layer thin film coatings of varying refractive indexes deposited on a transparent substrate. Edge filters are used more often than rugate filters because rugate filters are more expensive. Holographic filters, however, made by holographic means, typically have narrow rejection bandwidth, hence the name “notch” filters. The hologram media typically is fragile and requires special protection. In a number of commercially available holographic filters, a thin hologram media layer is sandwiched between two pieces of glass, and the edge is sealed with a special epoxy.
Compared with earlier interference edge filters, holographic notch filters had at least the advantage of a narrower rejection band, thus allowing observation of both Stokes and anti-Stokes Raman. Their edges also are steeper, allowing observation of Raman bands close to a laser line, down to less than 100 cm−1 Raman shift. The Raman transmission curve also is smoother and flatter, inducing less severe ripples to the observed spectrum. High quality interference filters, however, can match or exceed the performance of state-of-the-art holographic notch filters on the Stokes side of the Raman spectrum.
A second aspect of the Raman spectrometer disclosed and claimed in this document is the use of interference filters at low incident angles as beam combiners. The use of holographic filters at both large (45 degree) and small (less than 45 degree) incidence angles as beam-combiners to inject a laser beam into an optical path and to reject Rayleigh scattering has been suggested. The low (much less than 45 degree) incidence angle arrangement avoided the polarization effect of 45 degree dichroic mirror and allowed the observation of Raman lines very close to the laser frequency. However, it is known that holographic filters may induce fluorescence from the incident laser; they also are subject to damage from environmental factors such as moisture leaking into the hologram media. Performance of holographic filters made with current technology typically degrades over time. Interference edge filters and rugate filters, typically made of multi-layer hard oxide coatings, however, may be used for long periods of time without degradation. Also, because holographic filters are made individually, they cost much more than interference edge filters. If an interference edge filter or a rugate filter is used in a Raman spectrometer as a beam combiner, at low incidence angles less than 45 degrees, typically between 10 and 0 degrees, significant performance and low-cost advantages are achieved over holographic filters.